We plan to characterize a novel class of thymidine kinase (tk) negative mutants of Herpes simplex virus which lack a full-size tk polypeptide. Conclusive identification of the tk polypeptide and the relatedness of novel short polypeptides produced by these mutants will be accomplished by immunological techniques, partial purification, peptide analysis and hybrid arrested translation (HART). DNA from PBR322 containing a tk gene insert will be used as a probe in conjunction with "Southern" and "Northern" blotting techniques to determine whether some of the mutants are deletions and/or RNA processing mutations. Nonsense mutants will be identified by in vitro synthesis and suppression with yeast suppressor tRNAs and by microinjection techniques. Mammalian suppressor cell lines will be isolated by transformation of Ltk negative cells with DNA from a HSV-1 tk negative nonsense mutant and selection on HAT. tk ion colonies due to suppression rather than reversion, will be positively identified by infection with an HSV-2 tk virus containing an identical nonsense mutation and production of HSV-2 tk. In addition, a suppressor cell line will be constructed by co-transformation of a yeast suppressor tRNA gene with a tk nonsense mutation.